PROJECT SUMMARY/ABSTRACT TGF? and Wnt signaling pathways are involved in glaucoma. However, the cross-talk between them in the tra- becular meshwork (TM) and intraocular pressure (IOP) regulation is unclear. The goal of this project is to elu- cidate the role(s) of TGF? and Wnt signaling pathway cross-talk in POAG. Our objective is to determine the mechanisms and the biological consequence of this cross-talk in the TM and IOP regulation. The central hy- pothesis is that the cross-talk between the TGF? and Wnt pathways, which is mediated by a repressive tran- scriptional complex formed by Smad4 and ?-Catenin, regulates TM homeostasis and IOP. Our rationale is that our knowledge of the cross-talk will provide a novel therapeutic strategy in treating glaucoma by both in- hibiting the excessive TGF? signaling as well as elevating suppressed Wnt signaling in a number of POAG pa- tients. Guided by strong preliminary data, this hypothesis will be tested by pursuing 3 specific aims. SA#1: De- termine the effect of TGF? and Wnt signaling pathway cross-talk on IOP regulation; SA#2: Determine whether the Wnt pathway inhibits TGF?-induced pathological changes in TM cells; SA#3: Determine whether Smad4 and ?-Catenin form a protein complex, how the complex is formed, and its effect on signaling activities. In SA1, we will use a mouse model and human donor eyes to determine 1) whether there is a dose-dependent inhibition of TGF?2-induced OHT by Wnt3a in mouse eyes; 2) whether activation of the Wnt pathway affects TGF?2-induced OHT in Smad4 and ?-Catenin conditional knockout (KO) mouse eyes; and 3) whether activation of the Wnt pathway inhibits TGF?2-induced OHT in perfusion cultured human anteri- or segments. In SA2, we will determine 1) the genes that are cross-inhibited by each other?s pathway using RNA sequencing (RNAseq) and transgenic mouse TM (MTM) cell strains; 2) whether Wnt signaling inhibits TGF?2-induced formation of cross-linked actin networks in human TM (HTM) and transgenic MTM cells. In SA3, we will 1) determine the Smad4 and ?-Catenin protein complex in primary HTM cells using Co-IP, iTRAQ, and FRET; 2) identify the domains that are involved in Smad4-?-Catenin binding using computational analysis and mutational studies; 3) determine the effect of Smad4-?-Catenin interaction on promoter binding and transcriptional activities using gel electrophoresis mobility shift assay (EMSA), computational analysis, Chromatin immunoprecipitation (ChIP), luciferase assay, and qPCR. This project is significant, because upon the elucidation of this cross-talk, we will be able to manipulate both cell signaling pathways simultaneously. This strategy is expected to target the pathology of POAG in the TM that causes OHT. The approach is innova- tive, because 1) The cross-inhibition between the two pathways has never been demonstrated 2) We propose a unique molecular mechanism to define this cross-inhibition.; 3) A combination of RNA sequencing and trans- genic MTM cells; 4) A combination of in vivo (mice), ex vivo (human donor eyes), and in vitro (human and mouse primary TM cell cultures) models.